The recent discovery of new classes of small regulatory RNA species and the factors that control their biogenesis and function has revealed unexpected new mechanisms of gene regulation that affect normal development and, likely, cancer development. These discoveries have also led to the generation of powerful new tools for probing gene function in normal cells and in disease. This Project is focused on the investigation of the role of miRNAs in tumorigenesis and cell transformation as well as the use of RNAi to examine the function of genes in these processes. This will be investigated through a series of gene targeting experiments in the mouse directed at the mir-17approximately 92 cluster and its paralogs, which have been implicated in human tumorigenesis. These studies will also facilitate the discovery of the targets of these miRNAs. The let- 7 family of miRNAs, which have been reported to regulate Ras function, will be investigated using cell-based and in vivo methods. The role of miRNAs in tumorigenesis will be further tested by miRNA profiling experiments in a well-defined mouse model of lung cancer progression and through mutation of components of the miRNA processing machinery. Finally, this Project will seek to develop improved vectors and expression systems for constitutive and conditional shRNA expression in vivo. A specific application of this technology will be to test the function of a set of candidate tumor suppressor genes in lung cancer. Relevance statement: The elucidation of the molecular genetic events that underlie tumor development is critical for the effective treatment and prevention of cancer. This research is directed at understanding the function of a new class of regulatory molecules, termed miRNAs, in the development of cancer. The research will also lead to the development of powerful new tools to study cancer-relevant genes and pathways.